Effects Of Ripasudil On Proliferation And Migration Of Human Pulmonary Arterial Smooth Cells Induced By Platelet-derived Growth Factor-BB

Pulmonary arterial hypertension(PAH)is characterized by a persistent elevation of pulmonary artery pressure,leading to severe right heart failure and premature death without treatment.In recent years,significant progress has been made in the pathogenesis and pathophysiology of PAH.Pulmonary vascular remodeling,which is induced by pulmonary artery endothelial cell dysfunction and imbalance of smooth muscle cells proliferation and apoptosis,is as the main reason for PAH.The proliferation and migration of pulmonary artery smooth muscle cells(PASMCs)is the most significant.Rho/ROCK pathway is one of the important pathways to promote the proliferation and migration of PASMCs.Rho kinase is the key protein.PDGF-BB can activates Rho kinase in PASMC,which leads to a decrease in p27 and changes in cytoskeletal proteins,thereby promoting PASMC proliferation and migration.PAH is still an untreatable chronic disease.Because of the complexity of the pathogenesis,the current treatment is not always effective,so more new medicines are needed.For the past few years,many studies worldwide show that Rho kinase inhibitors can significantly inhibit the proliferation and migration of PASMCs,improve pulmonary vascular remodeling and reduce pulmonary arterial pressure,and have become a new target for the treatment of PAH.Ripasudil is a new type of Rho kinase inhibitor,which is highly selective for Rho kinase.It is currently used to treat glaucoma and ocular hypertension,but there is no relevant research in the treatment of pulmonary hypertension.Objective:This study aims to investigate the effects of ripasudil on proliferation and migration of human pulmonary arterial smooth cells(HPASMCs)induced by platelet-derived growth factor(PDGF)-BB and the mechanisms underlying.Methods:Cultured HPASMCs were divided into four groups:control group,PDGF-BB-treated group,PDGF-BB and ripasudil-treated group and ripasudil-treated group.CCK-8 was applied to investigate cell viability and EdU assay was used to evaluate the proliferation of HPASMCs.Transwell assay was employed to examine cell migration.The expression of matrix metalloproteinase-2(MMP-2)were determined by real-time PCR and western blot.The levels of phosphorylated myosin phosphatase target subunit 1(MYPT1),extracellar regulated protein kinases 1/2(ERK1/2),p38,and protein kinase B(PKA/Akt)were detected by western blotResults:CCK8 and EdU incorporation assay showed that different concentration of ripasudil(0.01,0.1,1,10 ?mol/L)was not toxic to the cells,PDGF-BB(20 ng/ml)significantly increased the cell viability and proliferation of HPASMCs after 48 hours,while treatment with 10 ?mol/L ripasudil could significantly inhibit PDGF-BB induced HPASMCs cell viability and proliferation.Transwell migration chamber assay showed that PDGF-BB significantly induced HPASMCs migration,which could be inhibited by ripasudil.RT-PCR and Western blot showed that ripasudil decreased the expression of MMP-2 mRNA and protein induced by PDGF-BB,significantly inhibit the increased phosphorylation level of MYPT1,ERK1/2,p38 and Akt induced by PDGF-BBConclusions:Ripasudil significantly inhibited PDGF-BB-induced proliferation and migration of HPASMCs,which might be attributed to the inhibition of phosphorylation of MYPT1,ERK1/2,p38 and Akt and decrease of MMP-2.Ripasudil,a Rho kinase inhibitor,might be a potential therapeutic option in PAH.